2.4.1 Measurement with crude Tmm
The reaction mixture was incubated for 15 min with 0.1 mM NADPH and 0.025 mM FMN and 20 µl of Tmm crude enzyme mix in the Tris-NaCl buffer. The reaction starts after adding trimethylamine and is quenched after 30 min by adding 0.1% TFA.
The amount of NADPH consumed from the reaction mix is calculated by monitoring the absorbance at 340 nm. And the amount of trimethylamine consumed was monitored by taking a 500 µl aliquot from the quenched reaction using trimethylamine-picrate formation. In another 500 µl aliquot, the amount of TMAO formed is calculated. From Table 2, the observed activity in the crude sample is higher in the case of NADPH+ assay. On checking the activity after each purification step, the activity values become comparable, proving that NADPH+-dependent assays are unreliable for crude samples.
Table 2 : Tmm enzyme assay and product quantification